The long-term objective of the proposed research is to elucidate the role of the Ca2+/lipid-dependent protein kinase C in photoreceptors. Specifically, the role of protein kinase C in rod outer segment structure and function will be addressed. Protein kinase C plays a critical role in the processing of extracellular information in a wide variety of signalling systems. Phosphorylation of target proteins results in alteration of both cell structure and function, while phosphorylation of receptors results in desensitization to incoming signals. The proposed research will examine the function and mechanism of action of this ubiquitous effector molecule in rod outer segments. Three specific aims will be addressed: 1] Function of Protein Kinase C in Rod Outer Segments: The hypothesis that protein kinase C is involved in phototransduction by phosphorylating rhodopsin will be tested. Preliminary results have revealed that treatment of retinas with phorbol esters, protein kinase C activators, results in a marked change in the light-dependent phosphorylation of rhodopsin. The proposed research will explore the mechanism by which protein kinase C affects the phosphorylation state of rhodopsin. In addition, a combination of in situ and in vitro phosphorylation experiments will be undertaken to identify other physiological substrates of protein kinase C in rod outer segments. 2] Characterization of Protein Kinase C from Bovine Retina: Biochemical and biophysical studies will address the mechanism of action of retinal protein kinase C. The hypothesis that retinal protein kinase C represents a unique isozyme will be investigated. Additionally, monoclonal antibodies to retinal protein kinase C will be generated. 3] Subcellular Distribution of Protein Kinase C in Dark- and Light- Adapted Rod Outer Segments: The distribution of protein kinase C isozymes in the retina (especially subcellularly in the photoreceptors) will be determined. The possibility that protein kinase C translocates between inner and outer segments, or from the cytosol to the membrane, in response to light, will be explored. Immunochemistry and subcellular fractionation studies will be undertaken on dark and light-adapted retinas and rod outer segments. The abundance of protein kinase C in rod outer segments, its ability to phosphorylate rhodopsin and the ubiquitous role of the enzyme in both signal transduction and receptor desensitization suggest that it may play a central role in photoreceptor function. Elucidating the role of protein kinase C in rod outer segments is central to a complete understanding of phototransduction and the cell biology of photoreceptors.